大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。     

大鼠松果体Clock基因和芳烷脘N-乙酰基转移酶基因的昼夜节律性表达及光照影响

探讨12h光照、12h黑暗交替(12h-light：12h-dark cycle,LD)及持续黑暗(constant darkness,DD)光制下松果体Clock基因和芳烷脘N-乙酰基转移酶基因(arylalkylamine N-acetyltransferase gene,NAT)是否存在昼夜节律性表达及其光反应变化。Sprague-Dawley大鼠在LD和DD光制下分别被饲养4周(n=36)和8周(n=36)后,在一昼夜内每隔4h采集一组松果体组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点样品中Clock及NAT基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律。结果如下：(1)在DD或LD光制下,松果体Clock和NAT基因mRNA的表达均呈现夜高昼低的节律性振荡(P＜0．05)。(2)与DD光制下比较,LD光制下松果体Clock和NAT基因的表达振幅及峰值相的mRNA水平均降低(P＜0．05)。(3)在DD或LD光制下,Clock和NAT基因之间显示相似的节律性表达(P＞0．05)。结果表明,Clock和NAT基因在松果体中存在同步的内源性昼夜节律表达,光照作用可使其表达下调。     

The ultrastructure of the human fetal pineal gland

Summary The ultrastructure of the pineal gland of 18 human fetuses (crown-rump lengths 30–178 mm) was investigated.The pineal gland exhibits a pyramidal shape and consists of an anterior and posterior lobe. Only one parenchymal cell type, the pinealocyte, was observed. Few neuroblasts were seen between the pinealocytes and in the extended perivascular space. The pinealocytes possess all the organelles necessary for hormone synthesis. No specific secretory granule could be observed. The organ is abundantly vascularized and richly innervated. The morphology of the capillaries indicates the existence of a blood-brain barrier.The ultrastructure of the human fetal pineal gland suggests that the gland has a secretory function in early intrauterine life. Acknowledgements. The author is grateful to Mrs. Yael Balslev and Miss Inger Ægidius for their able technical assistance. This investigation was supported in part by The Carl and Ellen Hertz's foundation and the Johann and Hanne Weimann foundation.     

Functional diversity on synaptic plasticity mediated by endocannabinoids

Endocannabinoids (eCBs) act as modulators of synaptic transmission through activation of a number of receptors, including, but not limited to, cannabinoid receptor 1 (CB1). eCBs share CB1 receptors as a common target with Δ⁹-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana. Although THC has been used for recreational and medicinal purposes for thousands of years, little was known about its effects at the cellular level or on neuronal circuits. Identification of CB1 receptors and the subsequent development of its specific ligands has therefore enhanced our ability to study and bring together a substantial amount of knowledge regarding how marijuana and eCBs modify interneuronal communication. To date, the eCB system, composed of cannabinoid receptors, ligands and the relevant enzymes, is recognized as the best-described retrograde signalling system in the brain. Its impact on synaptic transmission is widespread and more diverse than initially thought. The aim of this review is to succinctly present the most common forms of eCB-mediated modulation of synaptic transmission, while also illustrating the multiplicity of effects resulting from specializations of this signalling system at the circuital level.     

A morphological study of the circadian cycle of the pineal gland of the rat

Summary To seek a morphological expression of circadian rhythm, we investigated cytologically pineal glands taken from rats every 2 to 4 h under a lighting regime of 12 h of illumination (6:00 to 18:00) and 12 h of darkness. The changes in the number of synaptic ribbons and ribbon fields was observed by electron microscopy. The number of these intracellular elements was greatest at 2:00 and the lowest at 14:00, the difference being statistically significant. There were no significant local differences in numbers with respect to the part of the pineal gland examined. The data are similar to those of Vollrath from the pineal gland of a guinea pig, and seem to confirm a circadian function in the pineal gland in mammals.Supported by grants-in-aid from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare     

Granulated vesicles in the pineal gland of the mouse

Summary Pineal glands of normal adult mice, 7 to 42 days after bilateral superior cervical gangliectomy and 5 and 16 hours after one dose of reserpine (10 mg/kg) were studied under the electron microscope. The architecture of the gland is basically similar to that of other mammalian pineal glands previously studied. Mouse pinealocytes are polymorphic cells with perivascular and intercellular processes. Its most prominent feature is the presence of abundant granulated vesicles with a mean diameter of 1100 Å and a dense core of about 800 Å intermingled with clear vesicles of similar size distributed throughout the cytoplasm and more concentrated in perivascular and intercellular processes. These processes were seen in continuity with the perykaryon and remained after bilateral superior cervical gangliectomy. Processes containing the plurivesicular component of adrenergic nerves situated in perivascular and intercellular spaces disappeared after bilateral superior cervical gangliectomy. Reserpine depleted small granulated vesicles of nerves but the larger ones of perikaryon and polar processes remained almost unchanged.The histochemical technique of Wood was positive for catechol- and indolamines in the nerves. The reaction was negative in the perikaryon and polar processes.The significance of these findings is discussed.This work has been supported by grants of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina and U.S. Air Force AF-AFOSR 67-0963 A.I am greatly indebted to Miss Haydee Agoff and Mr. Alberto Saenz for their skillful technical assistance.     

Lcg is a light-inducible and clock-controlled gene expressed in the chicken pineal gland

The circadian clock is an autonomous biological clock that is entrainable to environmental 24-h cycles by receiving time cues such as light. Generally, light given at early and late subjective night, respectively, delays and advances the phase of the circadian oscillator. We previously searched for the chicken pineal genes that are induced by light in a phase-dependent manner. The present study undertook cDNA cloning and characterization of a gene whose expression was remarkably up-regulated by light at late subjective night. The mRNA level of this gene exhibited robust diurnal change in the pineal gland, with a peak in the early (subjective) day under light-dark cycles and constant dark condition, and hence it was designated Lcg (Light-inducible and Clock-controlled Gene). Chicken Lcg encodes a coiled-coil protein composed of 560 amino acid residues. Among chicken tissues, the pineal gland and the retina exhibited relatively high expression levels of LCG. LCG was colocalized with gamma-tubulin, a centrosomal protein, when expressed in COS7 cells, and LCG is the first example of a clock-related molecule being accumulated at the centrosome. Coimmunoprecipitation of LCG with gamma-tubulin in the chicken pineal lysate suggests a link between the circadian oscillator and the centrosomal function.     

Temporal-spatial characterization of chicken clock genes: circadian expression in retina,pineal gland,and peripheral tissues

The molecular core of the vertebrate circadian clock is a set of clock genes, whose products interact to control circadian changes in physiology. These clock genes are expressed in all tissues known to possess an endogenous self-sustaining clock, and many are also found in peripheral tissues. In the present study, the expression patterns of two clock genes, cBmal1 and cMOP4, were examined in the chicken, a useful model for analysis of the avian circadian system. In two tissues which contain endogenous clocks--the pineal gland and retina--circadian fluctuations of both cBmal1 and cMOP4 mRNAs were observed to be synchronous; highest levels occurred at Zeitgeber time 12. Expression of these genes is also rhythmic in several peripheral tissues; however, the phases of these rhythms differ from those in the pineal gland and retina: in the liver the peaks of cMOP4 and cBmal1 mRNAs are delayed 4-8 h and in the heart they are advanced by 4 h, relative to those in the pineal gland and retina. These results provide the first temporal characterization of cBmal1 and cMOP4 mRNAs in avian tissues: their presence in avian peripheral tissues indicates they may influence temporal features of daily rhythms in biochemical, physiological, and behavioral functions at these sites.     

Effect of lithium on the melatonin production in the pineal gland of viscacha

Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β₁-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.     

Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland

Conclusion The circadian rhythm of melatonin synthesis in the pineal glands of various species has been summarized. The night-time elevation of melatonin content is in most if not all cases regulated by the change of N-acetyltransferase activity. In mammals, the N-acetyltransferase rhythm is controlled by the central nervous system, presumably by suprachiasmatic nuclei in hypothalamus through the superior cervical ganglion. In birds, the circadian oscillator that regulates the N-acetyltransferase rhythm is located in the pineal glands. The avian pineal gland may play a biological clock function to control the circadian rhythms in physiological, endocrinological and biochemical processes via pineal hormone melatonin.     

Demonstration of action-potential-producing cells in the rat pineal gland in vitro and their regulation by norepinephrine and nitric oxide

There is evidence that sympathetically innervated mammalian pineal glands contain cells that exhibit action potentials. It is unknown whether ex vivo pineal glands deprived of their nervous input are still capable of firing. In the present study, multiple-unit recordings from rat pineals revealed spontaneously active cell clusters with a mean firing frequency of 1.5 ± 0.3 Hz which could be abolished by tedrodotoxin. Regularly firing clusters showed no inherent periodicity in the minute range, whereas rhythmical clusters with periodically repeated bursts had period lengths of 12.6 min (day) and 9.5 min (night). Superfusion of norepinephrine reduced the firing frequency of both cluster types, or had no effect, and the rhythmical clusters became regular. The effects of norepinephrine appear to be mediated via β-adrenoceptors because isoproterenol, a β-agonist, had the same effect as norepinephrine and as the α-agonist phenylephrine was without effect. Evidence was obtained that the inhibitory effect of norepinephrine may be mediated by nitric oxide, which appears to affect the electrical discharge of the cells directly or indirectly through cGMP. It is discussed whether the spontaneously active pineal cells represent an intrapineal oscillator or are driven by an intrinsic oscillator which has yet to be defined. Accepted: 11 August 1998     

Chronic exposure to 60-Hz electric fields: Effects on pineal function in the rat

As a component of studies to search for effects of 60-Hz electric field exposure on mammalian endocrine function, concentrations of melatonin, 5-methoxytryptophol, and serotonin-Nacetyl transferase activity were measured in the pineal glands of rats exposed or sham-exposed at 65 kV/m for 30 days. In two replicate experiments there were statistically significant differences between exposed and control rats in that the normal nocturnal increase in pineal melatonin content was depressed in the exposed animals. Concentrations of 5-methoxytryptophol were increased in the pineal glands of the exposed groups when compared to shamexposed controls. An alteration was also observed in serotonin-N-acetyl transferase activity, with lower levels measured in pineal glands from exposed animals.     

松果体昼夜节律生物钟分子机制的研究进展

在各种非哺乳类脊椎动物中 ,松果体起着中枢昼夜节律振荡器的作用。近来 ,在鸟类松果体中相继发现了几种钟基因 ,如Per、Cry、Clock和Bmal等 ,其表达的时间变化规律与哺乳类视交叉上核 (SCN)的非常相似。钟的振荡由其自身调控反馈环路的转录和翻译组成 ,鸟类松果体和哺乳类SCN似乎具有共同的钟振荡基本分子构架 ;若干钟基因产物作为正向或负向调节子影响钟的振荡 ;昼夜性的控时机制同时也需要翻译后事件的参与。这些过程对钟振荡器的稳定性和 /或钟导引的光输入通路有着重要的调控作用     

Melatonin rhythms: Trekking toward the heart of darkness in the chick pineal

Chick pineal cells make melatonin rhythmically, even in culture. Light pulses have two effects on these cells: acute suppression of melatonin synthesis and phase shifts (entrainment) of the underlying pacemaker. The two effects use different mechanistic pathways: the first goes through cAMP, and the second goes through the clock. Both converge on serotonin N-acetyltransferase, whose gene has recently been cloned, with cAMP acting on mRNA levels, but primarily on, enzyme activity, and the clock acting primarily on mRNA levels. Aspects of calcium regulation, not yet well-understood, may impinge on both pathways. These cells also have a novel calcium channel and a novel photopigment.     

Chronic stress decreases the expression of sympathetic markers in the pineal gland and increases plasma melatonin concentration in rats

Chronic stress affects brain areas involved in learning and emotional responses. Although most studies have concentrated on the effect of stress on limbic-related brain structures, in this study we investigated whether chronic stress might induce impairments in diencephalic structures associated with limbic components of the stress response. Specifically, we analyzed the effect of chronic immobilization stress on the expression of sympathetic markers in the rat epithalamic pineal gland by immunohistochemistry and western blot, whereas the plasma melatonin concentration was determined by radioimmunoassay. We found that chronic stress decreased the expression of three sympathetic markers in the pineal gland, tyrosine hydroxylase, the p75 neurotrophin receptor and alpha-tubulin, while the same treatment did not affect the expression of the non-specific sympathetic markers Erk1 and Erk2, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, these results were correlated with a significant increase in plasma melatonin concentration in stressed rats when compared with control animals. Our findings indicate that stress may impair pineal sympathetic inputs, leading to an abnormal melatonin release that may contribute to environmental maladaptation. In addition, we propose that the pineal gland is a target of glucocorticoid damage during stress.     

Presence of immunoreactive neurophysins of a higher molecular weight in addition to the 10.000 form in the ovine pineal gland

Using an aqueous extraction followed by ultrafiltration through Amicon Diaflo membranes, two ovine pineal fractions were obtained, which contain immunoreactive neurophysin. The presence of neurophysin was monitored by radioimmunoassay, employing an antiserum raised against pituitary bovine neurophysin and selected because it reacts with neurophysins of many other mammals. From 50 g of wet ovine pineal glands 552 micrograms of immunoreactive neurophysins were obtained. About 5% of these immunoreactive neurophysins are eluted from three different Sephadex columns with an elution volume corresponding to Mr above 10,000 between bovine serum albumin and pituitary neurophysin. The remaining 95% of ovine immunoreactive pineal neurophysin (Mr 10,000) shares immunological and physico-chemical properties with highly purified bovine pituitary neurophysin used as a reference. From the results of gel filtration and affinity chromatography on LVP-Sepharose it was concluded that ovine pineal gland may contain a neurophysin precursor molecule in addition to the neurophysin Mr 10,000.